Epigenetic modification of hippocampal Bdnf DNA in adult rats in an animal model of post-traumatic stress disorder

https://doi.org/10.1016/j.jpsychires.2011.01.013Get rights and content

Abstract

Epigenetic alterations of the brain-derived neurotrophic factor (Bdnf) gene have been linked with memory, stress, and neuropsychiatric disorders. Here we examined whether there was a link between an established rat model of post-traumatic stress disorder (PTSD) and Bdnf DNA methylation. Adult male Sprague–Dawley rats were given psychosocial stress composed of two acute cat exposures in conjunction with 31 days of daily social instability. These manipulations have been shown previously to produce physiological and behavioral sequelae in rats that are comparable to symptoms observed in traumatized people with PTSD. We then assessed Bdnf DNA methylation patterns (at exon IV) and gene expression. We have found here that the psychosocial stress regimen significantly increased Bdnf DNA methylation in the dorsal hippocampus, with the most robust hypermethylation detected in the dorsal CA1 subregion. Conversely, the psychosocial stress regimen significantly decreased methylation in the ventral hippocampus (CA3). No changes in Bdnf DNA methylation were detected in the medial prefrontal cortex or basolateral amygdala. In addition, there were decreased levels of Bdnf mRNA in both the dorsal and ventral CA1. These results provide evidence that traumatic stress occurring in adulthood can induce CNS gene methylation, and specifically, support the hypothesis that epigenetic marking of the Bdnf gene may underlie hippocampal dysfunction in response to traumatic stress. Furthermore, this work provides support for the speculative notion that altered hippocampal Bdnf DNA methylation is a cellular mechanism underlying the persistent cognitive deficits which are prominent features of the pathophysiology of PTSD.

Introduction

Post-traumatic stress disorder (PTSD) is a debilitating anxiety disorder which develops in a subset of people after they experience emotional trauma (American Psychiatric Association, 1994). PTSD patients are commonly plagued by recurrent frightening thoughts and memories of the experience and suffer from a host of persistent physiological and behavioral sequelae that include altered sympathetic and hypothalamic-pituitary-adrenal (HPA) axis responsivity, chronic anxiety, exaggerated startle, and cognitive dysfunction (Johnsen and Asbjørnsen, 2008, Mittal et al., 2001, Moore, 2009, Nemeroff et al., 2006, Yehuda and LeDoux, 2007). The biological basis of the development of PTSD is not fully understood, but the symptoms of PTSD have been linked to abnormalities in the functioning of the hippocampus, medial prefrontal cortex and amygdala (Bremner, 2007, Bremner et al., 2008, Heim and Nemeroff, 2009).

As PTSD shows moderate heritability, the etiology of PTSD has been hypothesized to be a product of complex gene-environment interplay (Afifi et al., 2010, Koenen et al., 2008, Yehuda and Bierer, 2009). One way traumatic events could affect genes is through DNA methylation, an epigenetic mechanism that produces functional changes in the genome without changes to the DNA sequence. Whereas DNA methylation has long been recognized for its role in establishing gene expression patterns during cellular development and differentiation, recent work has provided compelling evidence that DNA methylation remains an active process in the developing, as well as mature, CNS (Champagne and Curley, 2009, Graff and Mansuy, 2008, Roth and Sweatt, 2009, Zhang and Meaney, 2010). The susceptibility of the genome to epigenetic modifications provides a layer of genetic regulation that is potentially sensitive to a lifetime of experiential and environmental factors. Thus, an epigenetic explanation for environmental contributions to cognitive health and risk for neuropsychiatric disorders continues to gain traction (Costa et al., 2009, McGowan and Szyf, 2010; Roth et al., 2009b, Tsankova et al., 2007).

The involvement of DNA methylation specifically in emotional trauma is suggested from a broad range of work on stress, memory and animal models of PTSD. For example, Chertkow-Deutsher et al. (2010) recently provided evidence of methylation of the Disks Large-Associated Protein (DIgap2) gene in the hippocampus of rats briefly exposed to predator-related stimuli. Related work has focused on the brain-derived neurotrophic factor (Bdnf) gene, which is seen as a central player in mediating the effects of stress on CNS function and psychopathology, including PTSD and major depressive disorder (Calabrese et al., 2009, Casey et al., 2009, Duman and Monteggia, 2006, Kauer-Sant’Anna et al., 2007, McEwen, 2008). For example, rats exhibiting a PTSD-like response to trauma have been shown to exhibit a significant down-regulation of Bdnf mRNA in the hippocampus which correlated with PTSD-like behavioral stress responses (Kozlovsky et al., 2007). A recent study has documented changes in histone acetylation at individual Bdnf promoters following fear conditioning in animals previously subjected to a bout of prolonged stress (Takei et al., 2010). Moreover, a series of studies has demonstrated isoform-specific epigenetic marking, including methylation of Bdnf DNA, in rats exposed to either early developmental stress or fear conditioning in adulthood (Lubin et al., 2008, Roth et al., 2009a). Finally, hippocampus-specific deletion of the Bdnf gene impaired learning, and more importantly, reduced extinction of conditioned fear (Heldt et al., 2007), two findings that resonate with cognitive and emotional abnormalities commonly observed in people with PTSD. Taken together, there is strong support for the hypothesis that an ideal genetic target linking clinical PTSD symptoms with animal models of PTSD is the Bdnf gene in the hippocampus.

We have therefore investigated whether there are changes in Bdnf DNA methylation and mRNA expression in an animal model of PTSD which incorporates psychosocial factors known to contribute to the development and expression of PTSD in humans (Zoladz et al., 2008). Specifically, rats were immobilized while they were inescapably exposed to a cat, which elicits intense fear in rats (Blanchard et al., 1990, Blanchard et al., 1998, Hubbard et al., 2004) and inhibits hippocampal functioning and enhances amygdaloid activity (Adamec et al., 2005, Diamond et al., 1999, Diamond et al., 2007, Mesches et al., 1999, Park et al., 2008, Vouimba et al., 2006), all of which are clinical features of PTSD (Bremner, 2007, Bremner et al., 2008). Cat exposure, in conjunction with immobilization, models the lack of control and sense of horror and helplessness during trauma that are commonly reported by PTSD patients (American Psychiatric Association, 1994). As a core symptom of PTSD is the repeated re-experiencing of the traumatic event, such that patients often feel as if the trauma is actually happening at the present as opposed to a mere recollection (Ehlers et al., 2004, Reynolds and Brewin, 1999), rats were given a second inescapable cat exposure episode 10 days after the first to provide them with a reminder of their traumatic experience. We acknowledge there is a difference in internal re-experiencing, as can be seen in many PTSD patients following a single traumatic exposure, and the actual re-experiencing component that we use on rats. Thus, this aspect of our model may be most relevant to those people who develop PTSD as a result of multiple traumatic experiences. Finally, since a lack of social support and stability have been shown to contribute to PTSD (Andrews et al., 2003, Boscarino, 1995, Ullman and Filipas, 2001), housing conditions of rats were continually disturbed and randomized over the entire psychosocial stress period. We have shown that this combination of a life-threatening stressor, re-experiencing of the trauma, and chronic social instability produces a broad array of physiological and behavioral sequelae in rats, such as increases in cardiovascular and corticosteroid reactivity, increased anxiety, exaggerated startle, and cognitive impairments, all of which are remarkably similar to symptoms expressed by PTSD patients (Zoladz et al., 2008, Diamond and Zoladz, 2010a).

In summary, an extensive body of research has identified the hippocampus and Bdnf as vulnerable targets to traumatic stress in clinical and preclinical studies on PTSD. The current work extends this research to provide the first assessment of epigenetic modifications of the Bdnf gene in the hippocampus of adult rats in a well-established psychosocial stress animal model of PTSD.

Section snippets

Animals

Experimentally naïve adult male Sprague–Dawley rats (225–250 g upon delivery) obtained from Charles River laboratories (Wilmington, MA) were used for the present experiment. The rats were pair-housed on a 12-h light/dark schedule (lights on at 0700) in standard Plexiglas cages (two per cage) with free access to food and water. The colony room temperature and humidity were maintained at 20 ± 1 °C and 60 ± 3%, respectively. Upon arrival, all rats were given 1 week to acclimate to the housing room

Growth rate and organ weights

Growth rates, expressed as grams per day (g/day), were calculated for all rats by dividing their total body weight gained during the course of the experiment by the total number of days in the experiment (i.e., 31 days). Rats in the stress group exhibited significantly lower growth rates (4.47 ± 0.22 g/day) than rats in the no stress group (5.65 ± 0.47 g/day), t = 2.29, p < 0.05. There was no significant difference between the adrenal gland weights of the two groups (raw weight: t = 0.14, p

Discussion

The role of neurotrophins, particularly BDNF, in the neurobiological mechanisms underlying stress-related and memory disorders has been widely emphasized in a broad range of clinical and preclinical research (Calabrese et al., 2009, Casey et al., 2009, Duman and Monteggia, 2006, Kauer-Sant’Anna et al., 2007, McEwen, 2008). In the present study, we explored the influence of a chronic psychosocial stress regimen which has been shown to produce PTSD-like effects in rats on methylation of the Bdnf

Contributors

DMD initiated this project. Authors TLR and DMD designed the study. PRZ performed the psychosocial stress regimen, and TLR performed the biochemical analyses. All authors took part in interpretation of the results. TLR wrote the first draft of the manuscript, and PRZ, DMD, and JDS edited the manuscript.

Role of funding sources

Funding was provided by: Merit Review and Career Scientist Awards from the Veterans Affairs Department to DMD; National Alliance for Research on Schizophrenia and Depression and Civitan International Awards to TLR; and, National Institutes of Health, the Rotary Clubs CART fund, and the Evelyn F. McKnight Brain Research Foundation to JDS. Funding sources had no further role in: study design; in the collection, analysis and interpretation of data; in the writing of the report; and, in the

Conflict of interest

The authors report no biomedical financial interests or potential conflicts of interest.

Acknowledgements

We thank Dr. Mei Han and the University of Alabama at Birmingham Genomics Core Facility for assistance in DNA sequencing.

References (87)

  • K.E. Dennis et al.

    Regional expression of brain derived neurotrophic factor (BDNF) is correlated with dynamic patterns of promoter methylation in the developing mouse forebrain

    Molecular Brain Research

    (2005)
  • R.S. Duman et al.

    A neurotrophic model for stress-related mood disorders

    Biological Psychiatry

    (2006)
  • M.S. Fanselow et al.

    Are the dorsal and ventral hippocampus functionally distinct structures?

    Neuron

    (2010)
  • J. Graff et al.

    Epigenetic codes in cognition and behaviour

    Behavioural Brain Research

    (2008)
  • R. Grassi-Oliveira et al.

    Low plasma brain-derived neurotrophic factor and childhood physical neglect are associated with verbal memory impairment in major depression–a preliminary report

    Biological Psychiatry

    (2008)
  • S. Hauck et al.

    Serum brain-derived neurotrophic factor in patients with trauma psychopathology

    Progress in Neuro-Psychopharmacology and Biological Psychiatry

    (2010)
  • D.T. Hubbard et al.

    Development of defensive behavior and conditioning to cat odor in the rat

    Physiology and Behavior

    (2004)
  • M. Joëls

    Functional actions of corticosteroids in the hippocampus

    European Journal of Pharmacology

    (2008)
  • G.E. Johnsen et al.

    Consistent impaired verbal memory in PTSD: a meta-analysis

    Journal of Affective Disorders

    (2008)
  • K. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method

    Methods

    (2001)
  • B.S. McEwen

    Central effects of stress hormones in health and disease: understanding the protective and damaging effects of stress and stress mediators

    European Journal of Pharmacology

    (2008)
  • P.O. McGowan et al.

    The epigenetics of social adversity in early life: Implications for mental health outcomes

    Neurobiology of Disease

    (2010)
  • C.B. Nemeroff et al.

    Posttraumatic stress disorder: a state-of-the-science review

    Journal of Psychiatric Research

    (2006)
  • M. Nibuya et al.

    Repeated stress increases catalytic TrkB mRNA in rat hippocampus

    Neuroscience Letters

    (1999)
  • A.M. Rasmusson et al.

    Downregulation of BDNF mRNA in the hippocampal dentate gyrus after re-exposure to cues previously associated with footshock

    Neuropsychopharmacology

    (2002)
  • M. Reynolds et al.

    Intrusive memories in depression and posttraumatic stress disorder

    Behaviour Research and Therapy

    (1999)
  • T.L. Roth et al.

    Lasting epigenetic influence of early-life adversity on the BDNF gene

    Biological Psychiatry

    (2009)
  • T.L. Roth et al.

    Epigenetic mechanisms in schizophrenia

    Biochimica et Biophysica Acta (BBA) – General Subjects

    (2009)
  • T.L. Roth et al.

    Regulation of chromatin structure in memory formation

    Current Opinion in Neurobiology

    (2009)
  • R. Yehuda et al.

    Response variation following trauma: a translational neuroscience approach to understanding PTSD

    Neuron

    (2007)
  • T. Aid et al.

    Mouse and rat BDNF gene structure and expression revisited

    Journal of Neuroscience Research

    (2007)
  • American Psychiatric Association

    Diagnostic and statistical manual of mental disorders: DSM-IV

    (1994)
  • B. Andrews et al.

    Gender, social support, and PTSD in victims of violent crime

    Journal of Traumatic Stress

    (2003)
  • A. Bird

    DNA methylation patterns and epigenetic memory

    Genes and Development

    (2002)
  • J.A. Boscarino

    Post-traumatic stress and associated disorders among Vietnam veterans: the significance of combat exposure and social support

    Journal of Traumatic Stress

    (1995)
  • T.W. Bredy et al.

    Histone modifications around individual BDNF gene promoters in prefrontal cortex are associated with extinction of conditioned fear

    Learning and Memory

    (2007)
  • J.D. Bremner et al.

    Structural and functional plasticity of the human brain in posttraumatic stress disorder

    Progress in Brain Research

    (2008)
  • M. Chahrour et al.

    MeCP2, a key contributor to neurological disease, activates and represses transcription

    Science

    (2008)
  • W.G. Chen et al.

    Depression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2

    Science

    (2003)
  • Y. Chertkow-Deutsher et al.

    DNA methylation in vulnerability to post-traumatic stress in rats: evidence for the role of the post-synaptic density protein Dlgap2

    The International Journal of Neuropsychopharmacology

    (2010)
  • D.C. Choi et al.

    Prelimbic cortical BDNF is required for memory of learned fear but not extinction or innate fear

    Proceedings National Academy of Science

    (2010)
  • A. Collins et al.

    Exercise improves cognitive responses to psychological stress through enhancement of epigenetic mechanisms and gene expression in the dentate gyrus

    PLoS ONE

    (2009)
  • E. Costa et al.

    GABAergic promoter hypermethylation as a model to study the neurochemistry of schizophrenia vulnerability

    Expert Review of Neurotherapeutics

    (2009)
  • Cited by (255)

    • Environmental Influence on Epigenetics

      2022, Handbook of Epigenetics: The New Molecular and Medical Genetics, Third Edition
    View all citing articles on Scopus
    View full text